Microtubules are intracellular filamentous structures present in all eukaryotic cells. As components of different organelles such as mitotic spindles, centrioles, basal bodies, cilia, flagella, axopodia and the cytoskeleton, microtubules are involved in many cellular functions including chromosome movement during mitosis, cell motility, organelle transport, cytokinesis, cell plate formation, maintenance of cell shape and orientation of cell microfibril deposition in developing plant cell walls. The major component of microtubules is tubulin, a protein composed of two subunits called alpha and beta. An important property of tubulin in cells is the ability to undergo polymerization to form microtubules or to depolymerize under appropriate conditions. This process can also occur in vitro using isolated tubulin.
Microtubules play a critical role in cell division as components of the mitotic spindle, an organelle which is involved in distributing chromosomes within the dividing cell precisely between the two daughter nuclei. Various drugs and pesticides prevent cell division by binding to tubulin or to microtubules. Anticancer drugs acting by this mechanism include the alkaloids vincristine and vinblastine, and the taxane-based compounds paclitaxel and docetaxel {see, for example, E. K. Rowinsky and R. C. Donehower, Pharmacology and Therapeutics, 52, 35-84 (1991)}. Other antitubulin compounds active against mammalian cells include benzimidazoles such as nocodazole and natural products such as colchicine, podophyllotoxin and the combretastatins. Benzimidazole compounds which bind to tubulin are also widely used anthelmintics {McKellar, Q. A. and Scott, E. W., J. Vet. Pharmacol. Ther., 13, 223-247 (1990)}. Anti-tubulin herbicides are described in xe2x80x9cThe Biochemical Mode of Action of Pesticidesxe2x80x9d, by J. R. Corbett, K. Wright and A. C. Baillie, pp. 202-223, and include dinitroanilines such as trifluralin, N-phenylcarbamates such as chlorpropham, amiprophos-methyl, and pronamide. Fungicides believed to act by binding to tubulin include zarilamide {Young, D. H. and Reitz, E. M., Proceedings of the 10th International Symposium on Systemic Fungicides and Antifungal Compounds, Reinhardsbrunn, ed by H. Lyr and C. Polter, 381-385, (1993)}, the benzimidazoles benomyl and carbendazim, and the N-phenylcarbamate diethofencarb {Davidse, L. C and Ishi, H. in xe2x80x9cModern Selective Fungicidesxe2x80x9d, ed. by H. Lyr, 305-322 (1995)}.
Due to the success of tubulin as a biochemical target for drugs and pesticides, there is considerable interest in discovering new compounds which bind to tubulin. Various cell-free methods are available for detecting such compounds. A common method involves measuring the ability of test compounds to inhibit the polymerization of isolated tubulin into microtubules in vitro {see for example, E. Hamel, Medicinal Research Reviews, 16, 207-231 (1996)}. In a second method, interactions of test compounds with isolated tubulin can be detected in binding assays by measuring the ability of the test compound to influence binding of a second tubulin-binding ligand, used as a probe. (The term xe2x80x9ctest compoundxe2x80x9d means a compound which one wishes to evaluate, i.e. to test, for its ability to affect tubulin). Typically, the probe is radiolabeled to enable binding to be measured. A test compound which binds to tubulin may influence binding of the probe by binding to the same site on the tubulin protein as the probe, and thus reduce the amount of probe which binds. Alternatively, binding may be influenced by means of an xe2x80x9callostericxe2x80x9d interaction in which the test compound binds to a different site from that of the probe and induces a conformational change in the tubulin protein which affects the binding site of the probe. Such an allosteric interaction may either increase or decrease binding of the probe. A third approach involves measuring the effect of test compounds on tubulin-associated guanosine triphosphatase activity {Duanmu, C., Shahrik, L. K., Ho, H. H. and Hamel, E., Cancer Research, 49, 1344-1348 (1989)}.
To screen large numbers of compounds by any of these methods is feasible at present only using tubulin from mammalian brain tissue, since it has not been possible to isolate sufficiently large amounts of purified tubulin from other sources. This limits the usefulness of these methods since many anti-tubulin compounds show great specificity with respect to their effects on microtubules from different sources. For example, the herbicides oryzalin and amiprophosmethyl inhibit the polymerization of plant tubulin but not brain tubulin, whereas colchicine is more than 100-fold more effective as an inhibitor of brain tubulin polymerization than of plant tubulin polymerization {Morejohn, L. C. and Fosket, D. E., xe2x80x98Tubulin from Plants, Fungi, and Protistsxe2x80x99, in xe2x80x9cCell and Molecular Biology of the Cytoskeletonxe2x80x9d, ed. by J. W. Shay, 257-329 (1986)}.
The present invention relates to the use of certain amide derivatives, known to inhibit the growth of eukaryotic cells, including fungal and plant cells {see, for example, U.S. Pat. Nos. 3,661,991, 4,863,940 and 5,254,584}. Said amides have now been found useful as probes in binding assays to screen compounds for antitubulin activity, a use which U.S. Pat. Nos. 3,661,991, 4,863,940 and 5,254,584 neither disclose nor suggest. While radiolabeled probes such as colchicine {see for example, M. H. Zweig and C. F. Chignell, Biochemical Pharmacology, 22, 2141-2150 (1973)} and vinblastine (see for example, R. Bai et al., Journal of Biological Chemistry, 265, 17141 (1990)} have been used extensively in binding assays using isolated tubulin, these compounds bind noncovalently to tubulin.
One advantage of the amide derivatives of this invention over existing antitubulin compounds in competitive binding assays results from their unique ability to bind covalently in a highly specific manner to tubulin, specifically to the beta-subunit of tubulin. (A covalent bond is a nonionic chemical bond characterized by the sharing of electrons by two atoms). In binding assays it is necessary to measure the amount of the probe which is bound to tubulin, and this generally involves separating the tubulin-bound probe from unbound probe. In the case of the amides, since binding is covalent, the tubulin-bound probe is chemically stable allowing easy separation from the unbound probe by methods such as filtration or centrifugation. This enables their use not only in assays using isolated tubulin but also in assays using whole cells, crude cell extracts, and partially purified tubulin preparations, thus obviating the need for isolated tubulin and enabling tubulin-binding assays to be carried out in many different types of cell or cell extract.
One aspect of the present invention involves use of amide probes in binding assays to screen large numbers of compounds in order to identify those compounds with antitubulin activity using whole cells, cell extracts or isolated tubulin. For example, test compounds which bind to plant or fungal tubulin may be detected in assays using plant or fungal cells, thus providing a means of detecting antitubulin compounds with herbicidal or fungicidal activity. Similarly, amide probes may be used to detect compounds which bind to tubulin in mammalian cells or cell extracts, thus providing a means of detecting antitubulin compounds with anticancer activity.
A second aspect of the current invention involves use of amide probes in binding assays to evaluate the sensitivity of a cell population to an antitubulin compound. For example, the current invention can be used to evaluate the sensitivity of a tumor cell population to an antitubulin drug such as paclitaxel, vincristine or vinblastine, thus providing a means of predicting drug sensitivity of a patient""s tumor at the time of diagnosis or relapse using cells isolated by biopsy, and consequently guiding selection of the optimal chemotherapy regimen. Frequently, treatment of neoplasms with a particular antitubulin drug results in resistance development due to a reduced accumulation of drug in the cell. The current invention also provides a method for determining sensitivity of such resistant cells to antitubulin drugs. Various types of in vitro drug sensitivity tests have been used to select drugs more likely to be effective against tumor cells of a particular patient prior to their in vivo application {Cortazar, P. et al., Clinical Cancer Research, 3, 741-747 (1997), Arps, H. et al., Int. J. Immunotherapy, III, 229-235 (1987)}. Such assays typically involve cell culture of the isolated tumor cells or xenotransplantation using transplant-bearing mice, and require several days to multiple weeks to obtain results. In the current invention, the sensitivity of isolated tumor cells to antitubulin drugs can be determined by measuring the ability of said antitubulin drugs to influence binding of an amide probe to the cells, cell extracts or isolated tubulin. Since this method does not require culture of the isolated cells, it can provide sensitivity data within a few hours allowing drug sensitivity to be determined more rapidly.
A third aspect of the present invention involves another approach to the use of amide probes in binding assays to evaluate sensitivity of eukaryotic cells to pesticides or drugs which act by binding to tubulin. Specifically, this approach is useful in resistance monitoring for antitubulin pesticides or drugs to detect cells which show altered sensitivity to said antitubulin pesticides or drugs due to modifications in tubulin. Resistance to antitubulin compounds due to modifications in tubulin have occurred in fungal pathogens {Davidse, L. C. and Ishi, H. in xe2x80x9cModern Selective Fungicidesxe2x80x9d, ed. by H. Lyr, 305-322 (1995)}, algae {James, S. W. et al., Journal of Cell Science, 106, 209-218 (1993)} and helminths {Beech, R. N. et al., Genetics, 138, 103-110 (1994)}. Resistant cells containing modified tubulin may show a difference in binding affinity for amides, allowing amide probes to be used in binding assays to detect such mutants. Such an assay can be carried out by comparing the rate of binding of an amide probe to cells or extracts of cells previously exposed to the antitubulin pesticide or drug with the rate of binding to untreated control cells or cell extracts.
For example, benzimidazole and thiophanate fungicides such as benomyl (methyl 1-(butylcarbamoyl)benzimidazol-2-ylcarbamate), fuberidazole (2-(2xe2x80x2-furyl)benzimidazole), thiabendazole (2-(4-thiazolyl)benzimidazole), carbendazim (methyl benzimidazol-2-ylcarbamate), thiophanate-methyl (1,2-bis(3-methoxycarbonyl-2-thioureido)benzene, and thiophanate (1,2-bis(3-ethoxycarbonyl-2-thioureido)benzene are known in the art for use against plant pathogenic fungi. However, the use of benzimidazole and thiophanate fungicides over a period of time can result in the development of fungal strains having reduced sensitivity to these fungicides, whereby the fungicides are much less effective in controlling a particular fungal disease. Such xe2x80x9cresistantxe2x80x9d fungi when isolated as pure cultures typically are from 10-fold to  greater than 1,000-fold less sensitive to benzimidazoles and thiophanates than fungi from locations which have not been exposed to these fungicides. Moreover, fungi which develop reduced sensitivity to one benzimidazole or thiophanate fungicide frequently also show reduced sensitivity to other benzimidazole or thiophanate fungicides. The N-phenylcarbamate fungicide diethofencarb is used commercially to control benzimidazole-resistant fungi such as Botrytis cinerea. However, its use has led to the development of fungal strains resistant to both benzimidazoles and diethofencarb. Current methods to detect fungal strains resistant to benzimidazoles, thiophanates or diethofencarb are labor-intensive and time-consuming. Some methods involve isolation of pure test cultures followed by in vitro assays of mycelial growth using fungicide-amended agar plates, or in vivo assays involving fungicide-treated leaves. Alternatively, slide germination tests of spores may be carried out in the presence of fungicide. Fungal strains which are resistant to diethofencarb and/or benzimidazoles and thiophanates typically contain modified tubulin proteins {see for example, Koenraadt, H. et al., Phytopathology, 82, 1348-1354 (1992) and Yarden, O. and Katan, T., Phytopathology, 83, 1478-1483 (1993)}. Benzimidazole-resistant, diethofencarb-sensitive fungal strains typically show enhanced sensitivity to amide derivatives of the present invention, whereas benzimidazole-resistant, diethofencarb-resistant fungal strains typically show reduced sensitivity. While not wishing to be bound by theory, it is believed that amide probes can be used in binding assays to differentiate benzimidazole-resistant, diethofencarb-sensitive fungal strains which show enhanced ability to bind amide probes in assays using whole cells or cell extracts, or benzimidazole-resistant, diethofencarb-resistant fungal strains which show reduced ability to bind amide probes, from strains which are not resistant. Such assays may be less labor-intensive and time-consuming, and may also provide information as to whether the resistance mechanism involves a change in tubulin. Information about the mechanism of resistance may be useful in designing a resistance management strategy.
A fourth aspect of the present invention involves the use of amide probes in binding assays to detect and quantitate tubulin in cells or cell extracts. Tubulin is the subject of intense research due to its success as a target for drugs and pesticides and its important cellular functions. In such studies it is often desirable to detect and quantitate tubulin in cells or cell extracts. At present this is accomplished by various immunoassays {D. Thrower et al., Methods in Cell Biology, vol. 37, pp. 129-145 (1993)}, sodium dodecyl sulfate polyacrylamide gel electrophoresis {B. M. Spiegelman et al., Cell, vol. 12, pp. 587-600 (1977)}, binding to DEAE-cellulose {J. C. Bulinski et al., Analytical Biochemistry, vol. 104, 432-439 (1980)}, or by measuring colchicine-binding activity {Wilson, L., Biochemistry, vol. 9, pp. 4999-5007 (1970)}. Amide probes offer an alternative method to detect and quantitate tubulin based on measurement of amide-binding activity. Use of amide probes obviates the need for antibodies against tubulin, provides a simpler and more rapid method than either sodium dodecyl sulfate polyacrylamide gel electrophoresis or binding to DEAE-cellulose, and is applicable to measurement of tubulin levels in a variety of cells such as plant or fungal cells which are not sensitive to colchicine.
Various methods known to those with skill in the art can be used to detect the binding of amide probes to tubulin in assays using whole cells, crude cell extracts, partially purified tubulin preparations or isolated tubulin. This may, for example, involve the use of radiolabeled or fluorescent amide probes. Typically, the probe is incubated with cells, cell extracts or tubulin preparations and the amount of bound probe is determined following its separation from unbound probe by various separation techniques or a combination of such techniques. Separation techniques include, but are not limited to, centrifugation, chromatography, phase separation, precipitation of either the bound or unbound probe, or adhesion of either the bound or unbound probe to a solid substrate. Scintillation proximity assay technology (U.S. Pat. No. 4,568,649) provides another potential method to measure binding of the probe to tubulin. Yet another method involves detection of amide-bound tubulin, or amide-bound peptides released from tubulin by chemical or enzymatic treatment, by mass spectroscopy techniques.
One embodiment of this invention is a method for carrying out binding assays, which are useful for screening test compounds for antitubulin activity, comprising (i) incubating eukaryotic cells, cell extracts or isolated tubulin with the said test compound, (ii) adding an amide probe to the eukaryotic cells, cell extracts or isolated tubulin either simultaneously with the addition of the said test compound or subsequent to the addition of the said test compound and measuring the rate of binding of the said amide probe to tubulin in the eukaryotic cell, cell extract or isolated tubulin sample, and (iii) determining the antitubulin activity of the said test compound as indicated by a reduction or enhancement of the rate of binding of the said amide probe to tubulin in the sample containing the said test compound relative to the rate of binding of the said amide probe to tubulin in a control sample lacking the said test compound; said method using the said amide that binds covalently to tubulin and that inhibits the growth of eukaryotic cells, said amide having the structural formula 
wherein
A is phenyl, pyridyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, (C3-C7)cycloalkyl, pyrimidinyl, quinolyl, isoquinolyl, naphthyl, pyridazinyl, pyrazinyl, benzothienyl, indolyl, benzofuranyl or benzyl; or phenyl, pyridyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, (C3-C7)cycloalkyl, pyrimidinyl, quinolyl, isoquinolyl, naphthyl, pyridazinyl, pyrazinyl, benzothienyl, indolyl, benzofuranyl or benzyl substituted with up to three substituents each independently selected from halo, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, (C2-C4)alkenyl, halo (C2-C4) alkenyl, (C2-C4)alkynyl, halo(C2-C4)alkynyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, (C1-C4)alkylthio, halo(C1-C4)alkylthio, nitro, xe2x80x94NR6R7, xe2x80x94CR8xe2x95x90NOR9, xe2x80x94NHCOOR10, xe2x80x94CONR11R12, and xe2x80x94COOR13; or when A is an unsaturated ring and is substituted with two or more substituents which are adjacent to one another, two of said substituents may form a fused 5, 6 or 7 membered ring containing up to two heteroatoms selected from oxygen, nitrogen, sulfur and phosphorous;
R1 and R2 are each independently a hydrogen atom, (C1-C4)alkyl, halo(C1-C4)alkyl, (C2-C4)alkenyl, halo(C2-C4)alkenyl, (C2-C4)alkynyl or halo(C2-C4)alkynyl, provided that both R1 and R2 are not a hydrogen atom;
R6 and R7 are each independently a hydrogen atom, (C1-C4)alkyl or (C1-C4)alkylcarbonyl;
R8 is a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl or (C2-C4)alkynyl;
R9 is a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl or (C1-C4)alkylcarbonyl;
R10, R11, R12 and R13 are each independently a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl or (C2-C4)alkynyl; and
X, Y and Z are each independently a hydrogen atom, halo, cyano, thiocyano or isothiocyano, provided that at least one of X, Y and Z is not a hydrogen atom; or
the optical enantiomers thereof.
A second embodiment of this invention is a method for evaluating the sensitivity of a test cell population to an antitubulin compound comprising (i) incubating cells, cell extracts or isolated tubulin of the said test cell population with the said antitubulin compound, (ii) adding an amide probe to the cells, cell extracts or isolated tubulin of the said test cell population either simultaneously with the addition of the said antitubulin compound or subsequent to the addition of the said antitubulin compound and measuring the rate of binding of the said amide probe to tubulin in the cells, cell extracts or isolated tubulin of the said test cell population, and (iii) determining the sensitivity of the said test cell population to the said antitubulin compound by comparing the ability of the said antitubulin compound to affect binding of the said amide probe in the cells, cell extracts or isolated tubulin of the said test cell population with its ability to affect binding of the said amide probe in cells, cell extracts or isolated tubulin from cell populations of known sensitivity to the said antitubulin compound, said method using the said amide that binds covalently to tubulin and that inhibits the growth of eukaryotic cells, said amide having the structural formula 
wherein
A is phenyl, pyridyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, (C3-C7)cycloalkyl, pyrimidinyl, quinolyl, isoquinolyl, naphthyl, pyridazinyl, pyrazinyl, benzothienyl, indolyl, benzofuranyl or benzyl; or phenyl, pyridyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, (C3-C7)cycloalkyl, pyrimidinyl, quinolyl, isoquinolyl, naphthyl, pyridazinyl, pyrazinyl, benzothienyl, indolyl, benzofuranyl or benzyl substituted with up to three substituents each independently selected from halo, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, (C2-C4)alkenyl, halo(C2-C4)alkenyl, (C2-C4)alkynyl, halo(C2-C4)alkynyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, (C1-C4)alkylthio, halo(C1-C4)alkylthio, nitro, xe2x80x94NR6R7, xe2x80x94CR8xe2x95x90NOR9, xe2x80x94NHCOOR10, xe2x80x94CONR11R12, and xe2x80x94COOR13; or when A is an unsaturated ring and is substituted with two or more substituents which are adjacent to one another, two of said substituents may form a fused 5, 6 or 7 membered ring containing up to two heteroatoms selected from oxygen, nitrogen, sulfur and phosphorous;
R1 and R2 are each independently a hydrogen atom, (C1-C4)alkyl, halo(C1-C4)alkyl, (C2-C4)alkenyl, halo(C2-C4)alkenyl, (C2-C4)alkynyl or halo(C2-C4)alkynyl, provided that both R1 and R2 are not a hydrogen atom;
R6 and R7 are each independently a hydrogen atom, (C1-C4)alkyl or (C1-C4)alkylcarbonyl;
R8 is a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl or (C2-C4)alkynyl;
R9 is a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl or (C1-C4)alkylcarbonyl;
R10, R11, R12 and R13 are each independently a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl or (C2-C4)alkynyl; and
X, Y and Z are each independently a hydrogen atom, halo, cyano, thiocyano or isothiocyano, provided that at least one of X, Y and Z is not a hydrogen atom; or
the optical enantiomers thereof.
A third embodiment of this invention is a method for carrying out binding assays, which are useful for resistance monitoring for antitubulin drugs or pesticides to detect cells which show altered sensitivity to said antitubulin pesticides or drugs due to modifications in tubulin, comprising (i) measuring the rate of binding of an amide probe to tubulin in a test sample of eukaryotic cells, cell extracts or isolated tubulin from a cell population of unknown sensitivity to the antitubulin drug or pesticide and (ii) determining resistance as indicated by a reduction or enhancement of the rate of binding of the said amide probe to tubulin in the said test sample relative to the rate of binding of the said amide probe to tubulin in a corresponding control sample of known sensitivity to the said antitubulin drug or pesticide; said method using the said amide that binds covalently to tubulin and that inhibits the growth of eukaryotic cells, said amide having the structural formula 
wherein
A is phenyl, pyridyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, (C3-C7)cycloalkyl, pyrimidinyl, quinolyl, isoquinolyl, naphthyl, pyridazinyl, pyrazinyl, benzothienyl, indolyl, benzofuranyl or benzyl; or phenyl, pyridyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, (C3-C7)cycloalkyl, pyrimidinyl, quinolyl, isoquinolyl, naphthyl, pyridazinyl, pyrazinyl, benzothienyl, indolyl, benzofuranyl or benzyl substituted with up to three substituents each independently selected from halo, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, (C2-C4)alkenyl, halo(C2-C4)alkenyl, (C2-C4)alkynyl, halo(C2-C4)alkynyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, (C1-C4)alkylthio, halo(C1-C4)alkylthio, nitro, xe2x80x94NR6R7, xe2x80x94CR8xe2x95x90NOR9, xe2x80x94NHCOOR10, xe2x80x94CONR11R12, and xe2x80x94COOR13; or when A is an unsaturated ring and is substituted with two or more substituents which are adjacent to one another, two of said substituents may form a fused 5, 6 or 7 membered ring containing up to two heteroatoms selected from oxygen, nitrogen, sulfur and phosphorous;
R1 and R2 are each independently a hydrogen atom, (C1-C4)alkyl, halo(C1-C4)alkyl, (C2-C4)alkenyl, halo(C2-C4)alkenyl, (C2-C4)alkynyl or halo(C2-C4)alkynyl, provided that both R1 and R2 are not a hydrogen atom;
R6 and R7 are each independently a hydrogen atom, (C1-C4)alkyl or (C1-C4)alkylcarbonyl;
R8 is a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl or (C2-C4)alkynyl;
R9 is a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl or (C1-C4)alkylcarbonyl;
R10, R11, R12 and R13 are each independently a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl or (C2-C4)alkynyl; and
X, Y and Z are each independently a hydrogen atom, halo, cyano, thiocyano or isothiocyano, provided that at least one of X, Y and Z is not a hydrogen atom; or
the optical enantiomers thereof.
A fourth embodiment of this invention is a method for carrying out binding assays, which are useful for determination of tubulin content in eukaryotic cells, cell extracts or isolated tubulin, comprising (i) incubating the cells, cell extracts or isolated tubulin preparations of unknown tubulin content with an amide probe and (ii) comparing the rate of binding or maximum extent of binding of the said amide probe to tubulin in the sample of said unknown tubulin content with the rate of binding or maximum extent of binding of the said amide probe to tubulin in a sample of known tubulin content; said method using the said amide that binds covalently to tubulin and that inhibits the growth of eukaryotic cells, said amide having the structural formula 
wherein
A is phenyl, pyridyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, (C3-C7)cycloalkyl, pyrimidinyl, quinolyl, isoquinolyl, naphthyl, pyridazinyl, pyrazinyl, benzothienyl, indolyl, benzofuranyl or benzyl; or phenyl, pyridyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrrolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, (C3-C7)cycloalkyl, pyrimidinyl, quinolyl, isoquinolyl, naphthyl, pyridazinyl, pyrazinyl, benzothienyl, indolyl, benzofuranyl or benzyl substituted with up to three substituents each independently selected from halo, cyano, (C1-C4)alkyl, halo(C1-C4)alkyl, (C2-C4)alkenyl, halo(C2-C4)alkenyl, (C2-C4)alkynyl, halo(C2-C4)alkynyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, (C1-C4)alkylthio, halo(C1-C4)alkylthio, nitro, xe2x80x94NR6R7, xe2x80x94CR8xe2x95x90NOR9, xe2x80x94NHCOOR10, xe2x80x94CONR12, and xe2x80x94COOR13; or when A is an unsaturated ring and is substituted with two or more substituents which are adjacent to one another, two of said substituents may form a fused 5, 6 or 7 membered ring containing up to two heteroatoms selected from oxygen, nitrogen, sulfur and phosphorous;
R1 and R2 are each independently a hydrogen atom, (C1-C4)alkyl, halo(C1-C4)alkyl, (C2-C4)alkenyl, halo(C2-C4)alkenyl, (C2-C4)alkynyl or halo(C2-C4)alkynyl, provided that both R1 and R2 are not a hydrogen atom;
R6 and R7 are each independently a hydrogen atom, (C1-C4)alkyl or (C1-C4)alkylcarbonyl;
R8 is a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl or (C2-C4)alkynyl;
R9 is a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl, (C2-C4)alkynyl or (C1-C4)alkylcarbonyl;
R10, R11, R12 and R13 are each independently a hydrogen atom, (C1-C4)alkyl, (C2-C4)alkenyl or (C2-C4)alkynyl; and
X, Y and Z are each independently a hydrogen atom, halo, cyano, thiocyano or isothiocyano, provided that at least one of X, Y and Z is not a hydrogen atom; or
the optical enantiomers thereof.